The ability to efficiently modify the swine genome to generate genetically modified animals has the potential to provide a novel source of cells/tissues for clinical applications. Here we propose further development of the technology and its application to the development of genetically modified swine capable of high-level engraftment with human cells, and the testing and development of artificial organs. Specifically, we propose: Aim 1. To develop a genetically modified pig deficient in B cells, T cells, and NK cells (we will refer to this model as pSCID for porcine Severe Combined Immunodeficient from now on). Aim 1a. will accomplish this by ablating B cells and T cells using the toxin DTA. Aim 1b will utilize a responder line carrying a conditional DTA activated by Cre expression and an inducer line expressing Cre under the control of lymphoid specific promoters. Both lines can be easily propagated, and distributed, and the pSCID phenotype induced when the two lines are crossed. Aim2. To develop a pig hosting a human immune system. Aim 2a. We will utilize human/or swine bone marrow hematopoietic stem cells (HSC) to repopulate the immune system by injecting into a developing fetus prior to immune maturation. The porcine donors will be genetically labeled so that even in the absence of a pSCID we can complete this aim. Engraftment will be examined at one month, three months and six months of age by collection of bone marrow and peripheral blood. Aim 2b. Will examine the generated immune cells for the presence cell fusions between host and donor cells, presence of newly generated T cells using a T cell receptor excision circle assay (TREC), antibody receptor diversity by using oligonucleotide probes, proliferative responses using mixed leukocyte cultures, and examining novel T- cell specificities by immunizing with parvovirus, pseudorabies, and tetanus toxoid. Aim 3. To develop a transgenic pig that will facilitate engraftment and proliferation of human hepatocytes. Aim 3a will focus on generation of a transgenic swine carrying Cre-ERT2, a tamoxifen inducible Cre, driven by a liver-specific promoter. Crossing this line with the inducible DTA line generated in Aim 1 b, will allow depletion of the porcine hepatocytes upon tamoxifen addition. This will allow transplanted non-transgenic human hepatocytes to gradually replace the porcine hepatocytes. Aim 3b will focus on analyzing the resulting hepatocytes at multiple levels including expression of mature hepatocyte markers, induction of drug metabolizing enzymes, liver cytoarchitecture and presence/absence of cell fusions. This aim will allow us to determine the strengths and weaknesses of this xenotransplant organ regeneration approach so it can be used in the future to develop tissue and organs of greater complexity such as lung and cardiac tissue. PUBLIC HEALTH RELEVANCE: At completion of this proposal a number of unique transgenic swine will be available that can play a key role in the development and testing of artificial organs and tissue engineering but, in addition, have a broad range of uses in biomedical research. They can be used to study adoptive immunotherapy, normal and abnormal immune system development and function, study development and treatment for diseases such as leukemia, looking at disease progression and the development of vaccines for diseases such as AIDS, and study human tumor progression and treatment. In addition, the development of swine with livers largely composed of human hepatocytes will greatly facilitate pharmacological testing, the study of diseases such as hepatitis, development of models of liver-associated diseases such as cirrhosis, and eventual development of a human hepatocytes that can be transplanted back into humans.